Fig 1.
CPI-431-32 blocks both HIV-1 and HCV mono-infection.
CD4+ T-lymphocytes (0.5 x 106 cells) (triplicates) were exposed to HIV-1 JR-CSF (1 ng of p24) together with DMSO, the HIV-1 protease inhibitor nelfinavir, the HCV NS5A inhibitor daclatasvir and the two CypI ALV and CPI-431-32. Virus and drugs were washed away after 3 h and HIV-1 replication monitored for a period of 2 weeks every 3 days by quantifying amounts of virus in the cell culture supernatant by p24 ELISA (A). Same as A, except Huh7.5.1 hepatoma cells (0.5 x 106 cells) (triplicates) were exposed to HCV JFH-1 (1 ng of core) and replication monitored by ELISA for HCV core levels in supernatants (B), by RT-qPCR for HCV RNA levels in supernatants (C) or by colony forming assay for HCV infectious units in supernatants (D). Results are representative of two independent experiments.
Fig 2.
CPI-431-32 simultaneously blocks HIV-1 and HCV infections.
CD4+ T-lymphocytes (A) and Huh7.5.1 cells (B) (0.5 x 106 cells) (triplicates) were exposed to HIV-1 JR-CSF and HCV JFH-1. Drugs (2 μM) were added 3 h pre-viral exposure (top panels) or 3 days post-viral exposure (bottom panels). Viral replications were monitored by HIV-1 p24 and HCV core ELISAs. C. CD4+ T-lymphocytes (0.5 x 106 cells) (triplicates) were exposed to HIV-1 JR-CSF. Drugs were initially added 3 days post-viral exposure (2 μM) and then daily (0.5 μM). Viral replications were monitored by HIV-1 p24 and HCV core ELISAs. Results are representative of two independent experiments. D. Model for the HIV-1/HCV co-culture system.
Table 1.
CPI-431-32 inhibits the infection and replication of a panel of drug-resistant HIV-1 and HCV variants.
For HIV-1 infection, TZM cells were exposed to wild-type or HIV-1 drug-resistant variants together with increasing concentrations of DMSO, CsA, ALV or CPI-431-32. HIV-1 infection was quantified 48 h post-infection by measuring β-galactosidase activity in cell lysates. For HCV replication, Huh7.5 cells expressing wild-type or Luc-Con1 HCV drug-resistant variant replicons were exposed to increasing concentrations of DMSO, CsA, ALV or CPI-431-32. HCV replication was quantified 72 h post-infection by measuring luciferase activity in cell lysates. Data are expressed as EC50 that is the drug concentration necessary for the inhibition of HIV-1 infection (drug concentration expressed in μM) and HCV replication (drug concentration expressed in nM) in the absence of the drug by half.
Table 2.
CypA-HCV NS5A and CypA-HIV-1 Capsid Complex Inhibition Analyses.
Plates were coated with GST-CypA and blocked as we described previously [30]. Recombinant NS5A-His or capsid-His (1 ng/ml) was added to wells in binding buffer (20 mM Tris [pH 7.9], 0.5 M NaCl, 10% glycerol, 10 mM DTT, and 1% NP-40) together with increasing concentrations of DMSO, CsA, ALV or CPI-431-32 for 16 h at 4°C. Captured NS5A-His or capsid-His was subsequently detected using mouse anti-His antibodies and rabbit anti-mouse horseradish peroxidase phosphatase (HRP)-conjugated antibodies as we described previously [30]. After adding the OPD substrate, plates were read on a plate reader at 490 nm. Data are expressed as the concentration (IC50 nM) of CypI necessary to inhibit 50% of NS5A or capsid binding to CypA in the absence of drug.
Fig 3.
CPI-431-32 inhibits the isomerase activity of CypA.
CypA inhibition was assessed with the chymotrypsin-coupled isomerase inhibition assay. Representative data from one experiment is shown. Each symbol is the mean (± SE) of 4 replicate enzyme reactions. Mean IC50 values were 1.8 ± 0.6 nM (mean ± SD), 2.8 ± 0.4 nM, and 16.8 ± 2.3 nM for CPI-431-32, ALV and CsA, respectively.
Fig 4.
CPI-431-32 blocks HIV-1 reverse transcription and nuclear import.
TZM cells were infected with DNase-treated NL4-3 HIV-1 together with DMSO or CPI-431-32 (2 μM). Target cell DNA was isolated at the indicated times and was used to detect early (A) and late HIV-1 reverse transcripts (B). TZM cells were infected with NL4.3 in the presence or absence of CPI-431-32 (2 μM). Six hours post-infection, cells were fractionated and cytoplasmic and nuclear extracts analyzed by Western blotting (C). Results are representative of two independent experiments.
Fig 5.
CPI-431-32 blocks HCV-mediated double-membrane vesicle (DMV) formation.
T7 polymerase expressing Huh7.5.1 cells were transfected in triplicates with JFH-1 NS3-NS5B plasmid in the presence of DMSO or increasing concentrations of CPI-431-32 and analyzed by EM as well as by Image J and ITEM software for DMVs quantification as described previously [34]. Illustrating EM images of DMVs are presented with an amplification of 25,000x. Data are representative of two independent experiments.